Ultra Performance Liquid Chromatography and High Resolution Mass Spectrometry for the Analysis of Plant Lipids

Holistic analysis of lipids is becoming increasingly popular in the life sciences. Recently, several interesting, mass spectrometry-based studies have been conducted, especially in plant biology. However, while great advancements have been made we are still far from detecting all the lipids species in an organism. In this study we developed an ultra performance liquid chromatography-based method using a high resolution, accurate mass, mass spectrometer for the comprehensive profiling of more than 260 polar and non-polar Arabidopsis thaliana leaf lipids. The method is fully compatible to the commonly used lipid extraction protocols and provides a viable alternative to the commonly used direct infusion-based shotgun lipidomics approaches. The whole process is described in detail and compared to alternative lipidomic approaches. Next to the developed method we also introduce an in-house developed database search software (GoBioSpace), which allows one to perform targeted or un-targeted lipidomic and metabolomic analysis on mass spectrometric data of every kind

Metabolomic Response of Calotropis procera Growing in the Desert to Changes in Water Availability

Water availability is a major limitation for agricultural productivity. Plants growing in severe arid climates such as deserts provide tools for studying plant growth and performance under extreme drought conditions. The perennial species Calotropis procera used in this study is a shrub growing in many arid areas which has an exceptional ability to adapt and be productive in severe arid conditions. We describe the results of studying the metabolomic response of wild C procera plants growing in the desert to a one time water supply. Leaves of C. procera plants were taken at three time points before and 1 hour, 6 hours and 12 hours after watering and subjected to a metabolomics and lipidomics analysis. Analysis of the data reveals that within one hour after watering C. procera has already responded on the metabolic level to the sudden water availability as evidenced by major changes such as increased levels of most amino acids, a decrease in sucrose, raffinose and maltitol, a decrease in storage lipids (triacylglycerols) and an increase in membrane lipids including photosynthetic membranes. These changes still prevail at the 6 hour time point after watering however 12 hours after watering the metabolomics data are essentially indistinguishable from the prewatering state thus demonstrating not only a rapid response to water availability but also a rapid response to loss of water. Taken together these data suggest that the ability of C. procera to survive under the very harsh drought conditions prevailing in the desert might be associated with its rapid adjustments to water availability and losses

Proteome dynamics and early salt stress response of the photosynthetic organism <it>Chlamydomonas reinhardtii</it>

Abstract Background The cellular proteome and metabolome are underlying dynamic regulation allowing rapid adaptation to changes in the environment. System-wide analysis of these dynamics will provide novel insights into mechanisms of stress adaptation for higher photosynthetic organisms. We applied pulsed-SILAC labeling to a photosynthetic organism for the first time and we established a method to study proteome dynamics in the green alga Chlamydomonas reinhardtii, an emerging model system for plant biology. In addition, we combined the analysis of protein synthesis with metabolic profiling to study the dynamic changes of metabolism and proteome turnover under salt stress conditions. Results To study de novo protein synthesis an arginine auxotroph Chlamydomonas strain was cultivated in presence of stable isotope-labeled arginine for 24 hours. From the time course experiment in 3 salt concentrations we could identify more than 2500 proteins and their H/L ratio in at least one experimental condition; for 998 protiens at least 3 ratio counts were detected in the 24 h time point (0 mM NaCl). After fractionation we could identify 3115 proteins and for 1765 of them we determined their de novo synthesis rate. Consistently with previous findings we showed that RuBisCO is among the most prominent proteins in the cell; and similar abundance and turnover for the small and large RuBisCO subunit could be calculated. The D1 protein was identified among proteins with a high synthesis rates. A global median half-life of 45 h was calculated for Chlamydomonas proteins under the chosen conditions. Conclusion To investigate the temporal co-regulation of the proteome and metabolome, we applied salt stress to Chlamydomonas and studied the time dependent regulation of protein expression and changes in the metabolome. The main metabolic response to salt stress was observed within the amino acid metabolism. In particular, proline was up-regulated manifold and according to that an increased carbon flow within the proline biosynthetic pathway could be measured. In parallel the analysis of abundance and de novo synthesis of the corresponding enzymes revealed that metabolic rearrangements precede adjustments of protein abundance.</p

Targeted proteomics for Chlamydomonas reinhardtii combined with rapid subcellular protein fractionation, metabolomics and metabolic flux analyses.

peer reviewedIn the era of fast genome sequencing a critical goal is to develop genome-wide quantitative molecular approaches. Here, we present a metaproteogenomic strategy to integrate proteomics and metabolomics data for systems level analysis in the recently sequenced unicellular green algae Chlamydomonas reinhardtii. To achieve a representative proteome coverage we analysed different growth conditions with protein prefractionation and shotgun proteomics. For protein identification, different genome annotations as well as new gene model predictions with stringent peptide filter criteria were used. An overlapping proteome coverage of 25%, consistent for all databases, was determined. The data are stored in a public mass spectral reference database ProMEX (http://www.promexdb.org/home.shtml). A set of proteotypic peptides comprising Calvin cycle, photosynthetic apparatus, starch synthesis, glycolysis, TCA cycle, carbon concentrating mechanisms (CCM) and other pathways was selected from this database for targeted proteomics (Mass Western). Rapid subcellular fractionation in combination with targeted proteomics allowed for measuring subcellular protein concentrations in attomole per 1000 cells. From the same samples metabolite concentrations and metabolic fluxes by stable isotope incorporation were analyzed. Differences were found in the growth-dependent crosstalk of chloroplastidic and mitochondrial metabolism. A Mass Western survey of all detectable carbonic anhydrases partially involved in carbon-concentrating mechanism (CCM) revealed highest internal cell concentrations for a specific low-CO2-inducible mitochondrial CAH isoform. This indicates its role as one of the strongest CO2-responsive proteins in the crosstalk of air-adapted mixotrophic chloroplast and mitochondrial metabolism in Chlamydomonas reinhardtii

Experimental Setup and plants chosen.

<p>(a) Representative photo of the plants chosen for this experiment growing in its natural habitat in Saudi Arabia near to Jeddah. For this study representative species of similar size and performance were chosen. (b) Experimental set-up. At day 1 (control) leaves of three independent plants were harvested 1 h post-dawn, at midday and 1 h pre-dusk. One day later (Day 2), plants were watered at dawn and leaves were harvested 1 h post dawn, at midday and 1 h pre-dusk. Harvested leaves were frozen immediately in liquid –N and processes as described in Experimental procedures.</p

Clustered heatmap visualization of different lipid classes.

<p>Shown is the average abundance of several complex lipids visualized in a false-color heatmap at the three time points before and after watering ordered according to their presence in photosynthetic membranes, in cellular membranes or representing storage lipids.</p

Relative water content of leaves of <i>C. procera</i> plants taken at dawn, midday and one hour pre-dusk one day before watering and up to three days after watering.

<p>Relative water content of leaves of <i>C. procera</i> plants taken at dawn, midday and one hour pre-dusk one day before watering and up to three days after watering.</p

Principal Component Analysis (PCA) and ANOVA for metabolomic analysis of leaf samples from control and watered plants.

<p>(a) PCA (upper part) and ANOVA (lower part) for primary metabolites. (b) PCA (upper part) and ANOVA (lower part) for complex lipids. (c) PCA (upper part) and ANOVA (lower part) for secondary metabolites. Shown are always three independent samples per time point (dawn (1 hour post dawn/after watering), midday (6 hours after dawn/after watering) and pre-dusk (12 hours after dawn/after watering). Watered samples are shown in blue, non-watered in red. The lower part shows the results of a Bonferroni corrected ANOVA displaying the influence of treatment (watering) for all samples and of harvesting time for primary and secondary metabolites.</p

Boxplots of representative species of photosynthetic, structural and storage lipids.

<p>Boxplot-visualizations for a subset of complex lipids as determined for the three independent samples for the different time points and treatments as indicated on the x-axis of three replicates that were harvested at 1 h post-dawn, midday and 1 h pre-dusk for control and rehydrated plants.</p